Restrictions in the stem cell function of murine bone marrow grafts after ex vivo expansion of short-term repopulating progenitors.
We investigated the in vivo implications associated with the ex vivo expansion of 5-fluorouracil (5-FU) preactivated murine bone marrow (BM) grafts. Analysis of cultures established with BM cells collected 2 and 4 days after 5-FU treatment (2d and 4d 5-FU BM, respectively) and stimulated with IL-3 + IL-6 and IL-3 + SCF resulted in the generation of samples highly enriched for colony-forming units granulocyte/macrophage (CFU-GMs). This result was best shown in cultures established with 4d 5-FU BM and incubated for 3 days with IL-3 + SCF; in these samples up to 10% of the cellularity consisted of CFU-GMs. Analyses of the spleen colony-forming unit (CFU-S)12/CFU-S8 ratio revealed a continuous decline in this parameter during the expansion process, suggesting a predominant differentiation stimulus in the cultures. Transplantation of BM grafts into myeloablated recipients revealed a marked improvement in the short-term radioprotection capacity (30 days survival) of ex vivo expanded BM, which was most significant in the case of 3-day expanded grafts. In contrast to this finding, progressive impairment of the long-term radioprotection capacity of the grafts was found to be associated with the expansion process. Irrespective of the type of ex vivo manipulation used, a predominantly lymphohematopoietic repopulation by donor cells was observed in all recipients analyzed in the long term (4-7 months) posttransplantation. To investigate more thoroughly whether the repopulation ability of the grafts was modified to some extent during the ex vivo expansion process, BM competition assays were performed. The data obtained at 30 and 120 days posttransplantation indicated that under the best conditions assayed (4d 5-FU BM expanded for 3 days with IL-3 + SCF) almost no change in the competitive repopulation ability of the grafts was produced. However, when analysis was delayed to 300 days posttransplantation, a twofold reduction in the stem cell function of the expanded grafts was noted. Based on this data it is proposed that, under our experimental conditions, a significant expansion in the number of short-term repopulating progenitors is produced concomitantly with a differentiation stimulus of the culture, which moderately restricts the number and/or the longevity of the self-renewing stem cells.
Varas
F
F
Unidad de Biología Molecular y Celular, Centro de Investigaciones Energética Medioambientales y Tecnológicas, Madrid, Spain.
Bernard
A
A
Bueren
J A
JA
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D016026:Bone Marrow Transplantation / Q000502:physiology*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005472:Fluorouracil / Q000494:pharmacology*; D006085:Graft Survival / Q000502:physiology; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D013997:Time Factors
D007377:Interleukin-3; D015850:Interleukin-6; D005472:Fluorouracil